Vaccine 29 (2011) 4521–4533 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine A plant based protective antigen [PA(dIV)] vaccine expressed in chloroplasts demonstrates protective immunity in mice against anthrax  Jyotsna Gorantala a , Sonam Grover b,1 , Divya Goel a,1 , Amit Rahi a , Sri Krishna Jayadev Magani a , Subhash Chandra a,2 , Rakesh Bhatnagar a,∗ a Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India b Molecular Technology Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India article info Article history: Received 2 September 2010 Received in revised form 11 March 2011 Accepted 22 March 2011 Available online 17 April 2011 Keywords: Protective antigen Domain IV Anthrax Transplastomic plants Lethal toxin Protection abstract The currently available anthrax vaccines are limited by being incompletely characterized, potentially reactogenic and have an expanded dosage schedule. Plant based vaccines offer safe alternative for vaccine production. In the present study, we expressed domain IV of Bacillus anthracis protective antigen gene [PA(dIV)] in planta (by nuclear agrobacterium and chloroplast transformation) and E. coli [rPA(dIV)]. The presence of transgene and the expression of PA(dIV) in planta was confirmed by molecular analysis. Expression levels up to 5.3% of total soluble protein (TSP) were obtained with AT rich (71.8% AT content) PA(dIV) gene in transplastomic plants while 0.8% of TSP was obtained in nuclear transformants. Further, we investigated the protective response of plant and E. coli derived PA(dIV) in mice by intraperitoneal (i.p.) and oral immunizations with or without adjuvant. Antibody titers of >10 4 were induced upon i.p. and oral immunizations with plant derived PA(dIV) and oral immunization with E. coli derived PA(dIV). Intraperitoneal injections with adjuvanted E. coli derived PA(dIV), generated highest antibody titers of >10 5 . All the immunized groups demonstrated predominant IgG1 titers over IgG2a indicating a polarized Th2 type response. We also evaluated the mucosal antibody response in orally immunized groups. When fecal extracts were analyzed, low sIgA titer was demonstrated in adjuvanted plant and E. coli derived PA(dIV) groups. Further, PA(dIV) antisera enhanced B. anthracis spore uptake by macrophages in vitro and also demonstrated an anti-germinating effect suggesting a potent role at mucosal surfaces. The antibodies from various groups were efficient in neutralizing the lethal toxin in vitro. When mice were challenged with B. anthracis, mice immunized with adjuvanted plant PA(dIV) imparted 60% and 40% protection while E. coli derived PA(dIV) conferred 100% and 80% protection upon i.p. and oral immunizations. Thus, our study is the first attempt in highlighting the efficacy of plant expressed PA(dIV) by oral immunization in murine model. © 2011 Elsevier Ltd. All rights reserved. Abbreviations: AP, alkaline phosphatase; AVA, anthrax vaccine adsorbed; BAP, benzyl amino purine; BC, before challenge titers; cfu, colony forming units; CTAB, cetyltrimethyl ammonium bromide; CT, cholera toxin; DMEM, Dulbecco’s modified eagle medium; EF, edema factor; ELISA, enzyme linked immunosorbent assay; HRP, horseradish peroxidase; HEPES, N-(2-hydroxyethyl) piperazine-N ′ -(2 ethanesulfonic acid); i.p., intraperitoneal; IPTG, isopropyl -d-1-thiogalactopyranoside; LeTx, lethal toxin; LF, lethal factor; MAP kinase, mitogen activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-5 diphenyltetrazolium bromide; MTD, mean time death; MWCO, molecular weight cut-off; NAA, -naphthalene acetic acid; NBT, nitroblue tetrazolium; (NTdIV Nu), domain 4 from nuclear transformed plants; PA(dIV), domain IV of protective antigen B. anthracis; PAGE, poly acrylamide gel electrophoresis; PBS, phosphate buffered saline; PGA, poly-d-glutamic acid; PMSF, phenyl methyl sulfonyl fluoride; PA, protective antigen; SDS, sodium dodecyl sulphate; SM, selection medium; TSP, total soluble protein; TMB, tetramethylbenzidine; RM, regeneration medium; UTR, untranslated region; WT, wild type.  Gen Bank accession numbers of data deposited; pET-28a-PA(dIV): HQ130722, pCAM1303PA(dIV): HQ130723, pCHV-RKB: HQ130724, pCHV-RKB-PA(dIV): HQ130725. ∗ Corresponding author at: School of Biotechnology, Jawaharlal Nehru University, New Delhi 110067, India. Tel.: +91 11 26704079; fax: +91 11 26717040. E-mail addresses: rakbhat01@yahoo.com, rakeshbhatnagar@mail.jnu.ac.in (R. Bhatnagar). 1 Both the authors have contributed equally. 2 Present address: Dept. of Pop. Med. and Sciences, Veterinary Medical Centre, Cornell University, Ithaca, NY, USA. 0264-410X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2011.03.082